Site-specific DNA-protein interactions play a central role in all aspects of biology. This proposal focuses on the dynamics in protein-DNA complexes, forging links between the internal dynamics and measures of affinity and specificity. Binding of proteins to proximal sites on DNA is often cooperative, and the DNA itself may mediate cooperativity. Thus, the dynamics of both protein and DNA may influence neighboring interactions. Using the highly specific interaction of EcoRV endonuclease with DNA as a model, we investigate two probes of the dynamics: (a) DNA sequence context around the recognition site, which likely affects the energetic cost of DNA bending and the optimization of positions throughout the complex; (b) Lu3+ ions that bind in the active sites as Mg2+ surrogates and neutralize electrostatic repulsion between protein and DNA. Both probes have large effects on binding (1900-fold range for context, 22,000-fold for addition of metal ions) and markedly enhance specificity. Both have profound effects on the heat capacity change ?CP associated with EcoRV-DNA binding. ?CP is a key hallmark of specific protein-DNA interactions, because a large decrease in the heat capacity (?CP<<0) occurs for specific complexes, but not nonspecific complexes. It thus reflects goodness of fit in a protein-DNA interface. Flanking triple variation does not cause changes in the structure of the EcoRV-DNA complex or the degree of surface desolvation, thus greatly simplifying the analysis. Thermodynamic data suggest that the effects on ?CP are likely due to differences in dynamics of the protein-DNA complex and are too large to be merely local; preliminary NMR studies of methyl relaxation support this. Our central hypotheses are that both these factors tune dynamic freedom in the complex and that restricted dynamics make a dominant contribution to a negative ?CP. Thermodynamic studies on EcoRV-DNA complexes will measure probe-dependent variation in free energy, enthalpy, entropy and ?CP. NMR dynamics studies will determine whether dynamic changes due to DNA context, ions and active-site mutations map to distinct regions. Methyl carbon relaxation and exchange dispersion measurements will be used to characterize local ps-ns and s-ms motions of the protein. DNA dynamics will be assessed using relaxation and exchange dispersion of 31P, 19F, 15N and 13C groups in DNA. This includes a novel use of DNA phosphorothioates for NMR dynamics studies. Generalized NMR order parameters and their temperature-dependences will be used to relate dynamics to thermodynamic differences in ?S and ?CP. This project will thus illuminate how metal ions constrain conformational fluctuations to those on the path to the catalytic transition state, a potentially critical factor in catalysis y a wide variety of phosphodiesterases and other metalloenzymes. This can have important implications for design of drugs targeting key enzymes of infectious, genetic or metabolic diseases.